A22-11 Fibroblast-directed Reprogramming via Meflin Overexpression and Il-33 Decoy Delivered by Lnp-mRNA Reshapes Pro-fibrotic Signaling and Immune Activation in Human Pulmonary Fibroblast Models

Abstract Rationale Fibroblasts are central mediators of tissue fibrosis, contributing to extracellular matrix deposition and dysregulated immune responses in chronic lung diseases such as idiopathic pulmonary fibrosis (IPF). Meflin (ISLR) marks a fibroblast subset associated with anti-fibrotic properties, limiting myofibroblast differentiation and collagen deposition. IL-33, a key alarmin released by damaged epithelial and endothelial cells, promotes type-2 inflammation (Th2) and pro-fibrotic fibroblast activation. The decoy IL-33 receptor (dST2) sequesters IL-33 and mitigates Th2 biased immune responses, thereby acting to limit activation of fibroblasts while balancing type-1 inflammation (Th1) and Th2 immune cell phenotype. Adjuva Bio is developing a fibroblast-directed cell-engineering platform using lipid nanoparticle encapsulated mRNA (LNP-mRNA) delivery to simultaneously overexpress Meflin and dST2, aiming to reprogram pathogenic fibroblast phenotypes and restore tissue homeostasis. Methods The LL29 IPF cell line was transfected with LNP-formulated mRNA encoding both Meflin and IL-33 decoy ST2. LNP-mRNAs were optimized for efficient fibroblast uptake and sustained protein expression. Readouts at 48-96 h post-transfection include Immuno-fluorescence staining and imaging for α-SMA after TGF-Beta treatment at 5 ng/ml. The IPF cell line was also co-cultured with human PBMCs to assess T cell activation (CD69, CD25) and T helper cell profile using immuno-staining and flow cytometry. Results LNP-mRNA delivery to LL29 IPF cell line achieved robust expression of Meflin and decoy ST2 for ≥72h. Meflin overexpression reduced α-SMA and COL1A1 expression compared with control LNP-GFP as measured by fluorescence microscopy, while dST2 expression decreased IL-33 induced Th2 responses as determined by immuno-staining for CD69, CD25, and intracellular IL-13 and IL-4 cytokines using flow cytometry. Conclusions We have demonstrated that reprogramming of pathogenic fibroblasts via LNP-mRNA delivery of Meflin and IL-33 decoy ST2 effectively modulates fibrotic signaling and reshapes the immune microenvironment in vitro. We aim to further optimize the LNP delivery platform to enhance lung fibroblast-specific tropism. This fibroblast-directed approach using tissue tropic LNPs represents a novel therapeutic strategy to restore tissue homeostasis in IPF and related fibro-inflammatory diseases, supporting future in vivo evaluation and potential clinical translation. This abstract is funded by: None